Pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated from ovine (sheep) hypothalami based on its ability to stimulate adenylate cyclase activity in rat anterior pituitary cell cultures (Miyata et al., Biochem Biophys Res Commun 164:567-574, 1989). PACAP exists as two α-amidated peptides with 38 (PACAP38; SEQ ID NO:1) or 27 (PACAP27; SEQ ID NO:2) amino acids. Both peptides have the same N-terminal 27 amino acids and are synthesized from the same prohormone. The sequence of PACAP38 is identical in all mammals and differs from the avian and amphibian orthologs by only one amino acid (Vaudry et al., Pharmacol Rev 52:269-324, 2000). PACAP is a member of the secretin/vasoactive intestinal peptide (VIP)/growth hormone-releasing hormone (GHRH) family, and PACAP27 has 68% sequence identity with VIP (SEQ ID NO:3). PACAP is most abundant in the brain and testis, but there are significant levels in other organs, including the pancreas, adrenals, thymus, spleen, lymph nodes, and duodenal mucosa (Vaudry et al., Pharmacol Rev 52:269-324, 2000). PACAP is synthesized as a preprohormone and is processed mainly by prohormone convertase 1, prohormone convertase 2 and prohormone convertase 4 (Li et al., Neuroendocrinology 69:217-226, 1999; Li et al., Endocrinology 141:3723-3730, 2000). The half-life of [125I]-PACAP38 in the bloodstream of rats following intravenous injection is 5-6 minutes (Banks et al., J Pharmacol Exp Ther 267:690-696, 1993). Members of the secretin/VIP/GHRH family are degraded in plasma mainly by aminodipeptidases, especially dipeptidyl peptidase IV (Zhu et al., J Biol Chem 278:22418-2223, 2003).
A PACAP-specific receptor, designated as the PAC1 receptor, has been cloned from several vertebrate species (Arimura, Jpn J Physiol 48:301-331, 1998; Vaudry et al., Pharmacol Rev 52:269-324, 2000). It is a G-protein-coupled receptor with seven putative membrane-spanning domains and belongs to a family of glycoprotein receptors that are coupled to multiple signal transduction pathways (Segre and Goldring, Trends Endocrinol Metab 4:309-314, 1993). PACAP binds not only to the PAC1 receptor with a high affinity, but it also binds to the VIP1 (VPAC1) and VIP2 (VPAC2) receptors with an affinity comparable to or greater than VIP. On the other hand, VIP binds to the PAC1 receptor with an affinity 1,000 times less than PACAP (Arimura, Jpn J Physiol 48:301-331, 1998). At least 10 splice variants of the rat PAC1 receptor have been cloned and each variant is coupled to distinct combinations of signal transduction pathways (Vaudry et al., Pharmacol Rev 52:269-324, 2000). The “second” messengers include adenylate cyclase, phospholipase C, mitogen-activated protein (MAP) kinases, and calcium. PACAP/VIP receptor can be coupled to Gαs and/or Gαi in different types of cells. PACAP/VIP receptors are expressed in many different types of normal and cancer cells, including the catecholamine-containing cells in the adrenal medulla and the sympathetic ganglia; microglia, astrocytes and some types of neurons in the central nervous system; and T- and B-lymphocytes, macrophages, neutrophils, and dendritic cells in the immune system (Vaudry et al., Pharmacol Rev 52:269-324, 2000). PACAP is a potent stimulator of catecholamine secretion from the adrenal medulla (Watanabe et al., Am J Physiol 269:E903-E909, 1995), but a potent inhibitor of the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-12 from activated macrophages (Ganea and Delgado, Crit. Rev Oral Biol Med 13:229-237, 2002). More pertinent to the present invention, PACAP stimulates the proliferation of C6 glioblastoma cells (Dufes et al., J Mol Neurosci 21:91-102, 2003), AR4-2J pancreatic carcinoma cells (Buscail et al., Gastroenterology 103:1002-1008, 1992) and MCF-7 breast cancer cells (Leyton et al., Breast Cancer Res Treat 56:177-186, 1999), but inhibits the proliferation of HEL myeloid leukemia cells (Hayez et al., J Neuroimmunol 149:167-181, 2004), SW403 colonic adenocarcinoma cells (Lelievre et al., Cell Signal 10:13-26, 1998) and multiple myeloma cells (Li et al., Regul Pept 145:24-32, 2008; see FIGS. 3 and 4).
Although PACAP was isolated during a screen for novel hypophysiotropic factors, it soon became apparent that it is a pleiotropic peptide (Arimura, Jpn J Physiol 48:301-331, 1998; Vaudry et al., Pharmacol Rev 52:269-324, 2000). The extraordinarily potent neuroprotective/neurotrophic properties of PACAP were investigated by several laboratories shortly after its isolation. The cytoprotective effects of PACAP and VIP have been studied much more extensively in the nervous system than in any other major organ of the body. The cell types that were protected by PACAP in various in vitro models include cerebellar granule cells, dorsal root ganglion cells, sympathetic ganglion cells, mesencephalic dopaminergic neurons, and basal forebrain cholinergic neurons (Arimura, Jpn J Physiol 48:301-331, 1998; Vaudry et al., Pharmacol Rev 52:269-324, 2000). PACAP also prevented the neuronal death induced by gp120, the envelope glycoprotein of the human immunodeficiency virus (HIV), in rat hippocampal neuron/glia co-cultures. The dose-response curve was bimodal, with peaks at 10−13 M and 10−10 M (Arimura et al., Ann NY Acad Sci739:228-243, 1994). The critical findings in this study have been confirmed by Kong et al. (Neuroscience 91:493-500, 1999), who used lipopolysaccharide as the neurotoxin in primary murine cortical neuron/glia co-cultures. The neuroprotective effect at 10−12 M was correlated with a significant reduction in the accumulation of nitrite in the culture medium. The neuroprotective effect of “low” (femtomolar) doses of PACAP in neuron/glia co-cultures was abolished by PD98059, a MAP kinase inhibitor, but the neuroprotective effect of “high” (nanomolar) doses of PACAP was not affected by PD98059 (Li et al., J Mol Neurosci 27:91-106, 2005). However, the neuroprotective effect of nanomolar doses of PACAP was abolished by Rp-cAMP, a protein kinase A inhibitor.
The drawbacks of using peptides for neuroprotection in the brain include their poor transport across the blood-brain barrier and their short half-life in the circulation after systematic administration. However, PACAP38 has been shown to be transported from the blood to the brain via a saturable mechanism (Banks et al., J Pharmacol Exp Ther 267:690-696, 1993). Therefore, PACAP38 was tested as a neuroprotectant in common in vivo preclinical models of heart attack and stroke. Four-vessel occlusion in the rat was used to model the consequences of a heart attack for the brain (transient global forebrain ischemia). Blood flow to the forebrain was interrupted for 15 minutes. Following the 15-minute occlusion, there was a significant reduction in the number of pyramidal cells in the CA1 field of the hippocampus after 7 days in vehicle-infused rats. The reduction in the number of pyramidal cells at day 7 post-occlusion was significantly reversed in the rats continuously infused intravenously with PACAP38 (Uchida et al., Brain Res 736:280-286, 1996). Middle cerebral artery occlusion (MCAO) in the rat was used to model a stroke (transient focal cerebral ischemia). The middle cerebral artery was occluded for 2 hours using the intraluminal filament technique. The continuous intravenous infusion of PACAP38 beginning at 4, 8 or 12 hours after the start of the transient MCAO resulted in a reduction of the infarct volume of approximately 51%, 22% or 12%, respectively, 48 hours after the start of the MCAO (Reglodi et al., Stroke 31:1411-1417, 2000). These observations suggest that small changes in the concentration of PACAP in the brain can alter the vulnerability of nerve cells to injury.
The neuroprotective effects of low concentrations of PACAP in the nervous system are indirect and are probably mediated by at least four distinct mechanisms. (1) PACAP is a potent anti-inflammatory peptide. It has been shown to inhibit the induction of inducible nitric oxide synthase (iNOS) in activated macrophages, to inhibit the production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-12 in activated macrophages, and to stimulate the production of the anti-inflammatory cytokine IL-10 in activated macrophages (Ganea and Delgado, Crit Rev Oral Biol Med 13:229-237, 2002). PACAP probably inhibits inflammation at multiple steps in the inflammatory cascade because it is an endogenous counter-regulator of the inflammatory process. PACAP is also an extraordinarily potent “deactivator” of activated microglial cells (Kong et al., Neuroscience 91:493-500, 1999; Delgado et al., Glia 39:148-161, 2002), which are the resident macrophage-like cells in the nervous system. (2) Femtomolar (10−15 M) concentrations of PACAP increase the levels of the mRNA for activity-dependent neurotrophic factor in murine neuron/glia co-cultures (David et al., Society for Neuroscience [33rd Annual Meeting], New Orleans, La., #38.1 [Abstract], 2003). Furthermore, the number of PAC1 receptors on “reactive” glial cells is increased following injury (Uchida et al., Brain Res 736:280-286, 1996). Brenneman et al. (Neuropeptides 36:271-280, 2002) had previously shown that femtomolar concentrations of PACAP stimulate the release of RANTES in astrocyte cultures and that immunoneutralization of RANTES reduces the neuroprotective effect of PACAP in neuron/glia co-cultures. (3) Yang et al. (J Pharmacol Exp Ther 319:595-603, 2006) have shown that femtomolar concentrations of PACAP inhibit microglial NADPH oxidase activity and extracellular superoxide levels in mesencephalic neuron/glia co-cultures. (4) Figiel and Engele (J Neurosci 20:3596-3605, 2000) have reported that PACAP increased the expression of the glutamate transporters GLT-1 and GLAST and increased the activity of the glutamate metabolizing enzyme glutamine synthetase in astrocytes. These effects of PACAP would be expected to decrease glutamatergic neurotransmission. The extensive studies about the cytoprotective properties of PACAP in the nervous system have provided a solid framework for studying the cytoprotective properties of PACAP in other organs.
Native PACAP has already been administered to normal human volunteers by investigators in at least four different laboratories (Chiodera et al., Neuroendocrinology 64:242-246, 1996; Filipsson et al., J Clin Endocrinol Metab 82:3093-3098, 1997; Doberer et al., Eur J Clin Invest 37:665-672, 2007; Murck et al., Am J Physiol 292:E853-E857, 2007) and to a patient with multiple myeloma under a U.S. Food and Drug Administration (FDA)-approved protocol (Li et al., Peptides 28:1891-1895, 2007). The only untoward effect reported was a transient flushing.
PACAP is an extraordinarily potent peptide in vitro. However, the usefulness of PACAP as a drug is limited by its very short half-life in the circulation following systemic administration due to both rapid proteolysis and rapid filtration by the kidney. Therefore, there is a need for PACAP analogs that are resistant to proteolysis and/or have reduced rates of filtration by the kidney.
Citation or discussion of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.